Hydrophobe-substituted bPEI derivatives: boosting transfection on primary vascular cells

Abstract

Gene therapy targeted to vascular cells represents a promising approach for prevention and treatment of pathological conditions such as intimal hyperplasia, in-stent and post-angioplasty restenosis. In this context, polymeric non-viral gene delivery systems are a safe alternative to viral vectors but a further improvement in efficiency and cytocompatibility is needed to improve their clinical success. Herein, a library of 24 branched polyethylenimine (bPEI) derivatives modified with hydrophobic moieties was synthesised, characterised and tested in vitro on primary vascular cells, aiming to identify delivery agents with superior transfection efficiency and low cytotoxicity. Low molecular weight PEIs (0.6, 1.2 and 2 kDa) were grafted with long (C18) and short (C3) aliphatic chains, featuring different unsaturation degrees and degrees of substitution. 0.6 kDa bPEI-based derivatives were generally ineffective in transfection on vascular smooth muscle cells (VSMCs), while among the other derivatives some promising vectors were identified. Forcing polyplexes on the cell surface by means of centrifugation invariably boosted transfection levels but increased cytotoxicity as well. Of note, a propionyl-substituted derivative (PEI2-PrA1, C3:0) was the most effective on both VSMCs and endothelial cells (ECs), with higher and more sustained gene expression in combination with markedly lower cytotoxicity with respect to the gold standard 25 kDa bPEI. In addition, a linoleoyl-substituted derivative (PEI1.2-LA6, C18:2) owing to its high efficiency in VSMCs and relative inefficacy in ECs, combined with tolerable cytotoxicity was proposed as a vector for specific VSMCs targeting.

Increasing Cell Seeding Density Improves Elastin Expression and Mechanical Properties in Collagen Gel-Based Scaffolds Cellularized with Smooth Muscle Cells

“…Altogether, these results suggest the use of higher SMC seeding densities (>1.5 × 10 6 cells/mL) for the preparation of collagen gel‐based vascular tissue models in order to speed up the maturation of the constructs and obtain structural and mechanical properties better mimicking those of native arteries. The combination of high seeding densities with other strategies to ameliorate engineered blood vessel properties such as co‐culture of cells, dynamic maturation by means of mechanical stimulation in bioreactors to reproduce the mechanical stimulation generated by blood flow and transfection of vascular cells with ECM‐relevant genes will possibly allow to rapidly obtain physiologically relevant constructs for vTE applications.…”

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

“…Because they are relatively safe, display easily tunable physico-chemical properties, can be produced in large quantities with high reproducibility and affordable costs, and show unlimited ferrying capacity [53][54][55], non-viral vectors are nowadays at the forefront of gene delivery [1]. The two major approaches to non-viral gene delivery involve the combination of NAs with transfection molecules, that is, cationic lipids (CLs; e.g., DOTAP, DOTMA) [56][57][58][59][60][61] in different forms, and cationic polymers (CPs; e.g., poly-L-lysines, polyethyleneimines (PEIs), poly(amidoamine)s, chitosans) [62][63][64][65][66][67]. The beauty of this approach lies in its simplicity, yet in its effectiveness.…”

“…Besides, they can display different chemistries and architectures [93]. CPs include commercially sourced materials ( Figure 2), such as poly-L-lysines (PLLs) [94,95], poly(ethyleneimines) (PEIs) [96,97], poly(amidoamines) (PAMAMs) [64,[98][99][100][101], poly [2-(dimethyamino)ethyl methacrylates] (PDMAEMs) [102], and chitosans (CSs) [103][104][105][106], used as received or reacted and functionalized [63,[107][108][109][110], and those purposely synthesized [111]. Of note, the efficacy of a transfectant strongly depends on some chemical and geometrical features.…”

“…Furthermore, other parameters herein disclosed, such as the volume ratio between the polyplex suspension and the culture medium used during transfection of adherent cells (hereinafter referred to as the polyplex volume:medium volume ratio), and the way complexes are dispensed to cells in culture ( Figure 6) have a striking effect on the transfection behavior of gene delivery vectors [62,63,108,113,125,128,[151][152][153][154]. Specifically, the lower the transfectant volume:medium volume ratio (e.g., ≤1:40, which corresponds to deliver ≤2.5 µL of complexes to cells cultured in a 96-multiwell plate format with 100 µL/well of medium), the greater the transfection efficiency of polyplexes (Figure 6a).…”

Fibronectin promotes elastin deposition, elasticity and mechanical strength in cellularised collagen-based scaffolds

“…Primary porcine aortic SMCs (PAoSMCs) were derived from the tunica media of healthy, fibrous plaque-free porcine arteries. PAoSMCs were isolated from 1 cm wide aortic rings obtained from segments of the aorta of adult pigs [40]. Aortic segments were collected in phosphate-buffered saline (PBS), placed in Dulbecco's Modified Eagle's Medium (DMEM, Life Technologies) supplemented with 1% penicillin/streptomycin solution (100 U/mL penicillin, 0.1 mg/mL streptomycin; Sigma) and then dissected.…”